E2A-PBX1 ChIP binding.

<p><b>A. Peak promoter sequence analysis</b>. Chromatin immunoprecipitation assays were performed to identify direct targets E2A-PBX1 using the human promoter array; HG18; (NCBI Build 36, Roche Nimblegen, Madison WI). A search was performed using the software WebMOTIFS and de novo motif finding a search for Transcription Factors that are likely to contact and regulate the binding sites identified by ChIP-chip. The 3 most significant transcription factor families (classes of transcription factors most likely to regulate the input sequences) are PBX, POU and STAT. <b>B. Positional binding of E2A-PBX1</b>. Among the 108 top hit ChIP genes, 76 genes have a single annotated transcription start site. The average intensity of ChIP pull-down is graphed along the promoter region in a smoothed plot. This histogram of the positions of maximal peak intensities (mean of 3 replicates) relative to the transcription start site (TSS) in target genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087602#pone.0087602.s007" target="_blank">Table S3</a>). Most target genes show a maximum probe intensity around −500 bp upstream of the TSS. Probe regions with multiple TSS were excluded from this analysis. The promoter coverage area was complete for all genes between −2200 and +500 by the TSS (solid line) with some additional coverage for some genes outside of that region (dotted line).</p

Expanded combined analysis of direct and functional E2A-PBX1 targets.

<p>The analysis was performed using the 102 direct targets identified by ChIP-chip analysis and the data set of the functional targets including the top 2000 genes up and down regulated after siRNA silencing of E2A-PBX1. Venn diagrams were generated and the interfaces of direct and functional E2A-PBX1 targets are depicted. 10 genes that were both direct targets and functionally down-regulated <b>(</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087602#pone-0087602-g005" target="_blank"><b>Figure 5A</b></a><b>)</b> and 10 that were both direct targets and functionally up-regulated <b>(</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087602#pone-0087602-g005" target="_blank"><b>Figure 5B</b></a><b>)</b> dependent on expression of E2A-PBX1 were identified. We are not able to address E2A-PBX1 as a functional repressor (for the 10 down regulated genes) or whether its depletion allowed accessibility to another transcriptional activator. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087602#pone-0087602-g005" target="_blank"><b>Figure 5C</b></a> Selected direct and functional E2A-PBX1 targets are highly expressed in primary E2A-PBX1 leukemias. Pediatric Cancer Genome Project. <a href="http://www.pediatriccancergenomeproject.org/site/accessed" target="_blank">http://www.pediatriccancergenomeproject.org/site/accessed</a> 14 Oct 2013. The expression of four genes UGT2B15, ASNS, WNT16 and HK2, that were among the direct and functional E2A-PBX1 targets in primary leukemia’s are shown. The expression of these genes was analyzed in Hyperdiploid leukemia’s (H), in E2A-PBX1 leukemia’s (E), TEL-AML1 (T) leukemia’s and BCR-ABL1 (B) leukemia’s are shown. The analysis of expression was performed using the gene expression tool and data available at St. Jude Children’s Research Hospital – (Washington University).</p

Identified ChIP-chip targets on expression array in E2A-PBX1 silenced vs control cells.

<p><b>A.</b> ChIP-chip analysis data and expression analysis data upon E2A-PBX1 silencing were combined to get an overview of the direct and functional targets of E2A-PBX1. siRNA to E2A-PBX1 was employed to silence E2A-PBX1. The MA plot depicts the expression array changes upon silencing E2A-PBX1. Genes are ordered on the x-axis on the basis of their expression in untreated 697 cells. The Y-axis displays the change in expression upon E2A-PBX1 silencing. Those genes that are direct hits of E2A-PBX1 by chromatin immunoprecipitation assays (108 genes) were plotted against the expression array changes upon silencing and are shown in red color and marked with a blue dot; grey dots indicate all other genes assessed by the array. <b>B.</b> Here we compare the changes in expression of the 108 E2A-PBX1 direct targets to the expression change of all the other genes (non-direct E2A-PBX1 targets) upon E2A-PBX1 silencing. The set of identified ChIP-chip targets show collective down-regulation trend in the E2A-PBX1 silenced samples compared to controls (p<1.63e-06).</p

Combined KEGG Pathway analysis of direct and functional E2A-PBX1 targets.

<p>The analysis was performed using Exploratory Gene Association Networks (EGAN) software tool. E2A-PBX1 direct (108 direct targets identified by ChIP-chip) and functional targets (122 significant differentially expressed genes between E2a-Pbx1 silenced samples and controls) and Pathways that might be regulated by them were visualized. Interfaces of direct and functional E2A-PBX1 targets are depicted. Magenta lines depict the connection of the genes to the direct target and/or functional target group; blue lines show the participation of the genes in KEGG pathways and brown lines show known interaction between genes connected.</p

The 9p21 deletion is not always targeting CDKN2A.

<p>Copy number alterations in a group of 22 t(1;19)⁺<i>E2A-PBX1</i> bone marrow samples are shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087602#pone-0087602-g003" target="_blank"><b>Figure 3A</b></a>. Patient samples were subjected to hierarchical clustering analysis; we found the 9p21 deletion to exist in four of the 22 patients we have analyzed, with one patient exhibiting a homozygous deletion, white color refers to no changes, red to gain and blue to loss. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087602#pone-0087602-g003" target="_blank"><b>Figure 3B</b></a>. Fine map of deletions in the 9p21 region showing the deletion endpoints among 8 patients. The commonly deleted region among 10 patients (with 45%, including 7 patients in this figure and the other 3 others with complete arm loss) is the interferon gene cluster telomeric to the <i>CDKN2A/B</i> locus.</p